User:Lori Wetmore/Sandbox 4

Background Information
ATP-binding cassette (ABC) transporters are a superfamily of integral membrane proteins that harness the energy of ATP binding and hydrolysis to drive the trans-membrane movement of a variety of small molecules. ABC transporters function as homodimers, in which ATP binding and hydrolysis occurs in two sites that the interface of the nucleotide binding domains (NBD), while the paired transmembrane domains (TMD) facilitate substrate transport. Substrates may be imported or exported, depending upon the structure of the transporter.

ABC transporters are of particular medical interest, as they may contribute to the pathogenicity and drug resistance of pathogenic bacteria. In humans and other eukaryotes, ABC transporters function to pump substrates across both the plasma membrane and internal membranes. Defects in ABC transporters can manifest as a variety of inheritable diseases, such as cystic fibrosis, anemia, retinal and neurological degeneration, and other transport defects. ABC transporters also highly expressed in some multi drug-resistant cancers, where they are involved in removing drugs from the cytosol.

ABC transporters can be subdivided into three categories: exporters, type I importers, and type II importers. In ABC importers, which have only been found in prokaryotes, the NBD and TMD are separate polypeptides; however, in the exporters, the NBD and TMD are fused. ABC exporters are incredibly conserved across all three domains of life, with the ATP bind cassette itself being far more conserved than the specific TMDs to which they are linked.

General ABC Structure


The ATP binding cassette is the most conserved part of an ABC transporter. All ABCs consist of two domains: a RecA-like domain, containing both the Walker A and Walker B motifs, and a helical domain, that contains a unique LSGGQ motif. The two domains are joined by flexible loops, one of which, the Q loop, mediates the interaction between the ABC and the TMD.

ABC transporters function as homodimers. The two ATP binding sites of an assembled transporter are at the interfaces of two ABC subunits, where the ATP interacts with the Walker A motif (yellow) on one subunit and the LSGGQ motif (pink) on the other. The Walker A motif has the sequence GxxGxGKST, in which the well-conserved lysine residue (shown in green), stabilizes the bound ATP by hydrogen bonding with the alpha and gamma phosphates. The residue shown in magenta is a highly conserved histidine from the nearby H loop. This histidine hydrogen bonds with the gamma phosphate of the bound ATP and plays an important role in ATP hydrolysis, necessary for the correct functioning of the transporter.

Essential to the role of ABC transporters is their ability to convert the energy of ATP binding and hydrolysis into the transmembrane motion of their substrates. This transfer of energy is accomplished by a specific series of conformational changes shared by all ABC transporters. The cycle begins in a ground state, after the NBDs have released ADP and Pi and are nucleotide free. At this time the substrate binding/extrusion site in the TMD faces the cytosolic side of the membrane. Subsequently, the transporter binds two ATP molecules, one at each of the ATP binding sites located at the interface between the NBDs. Binding of ATP draws the NBDs into a closed conformation. The motion of the NBDs is coupled to the TMDs via highly conserved coupling helices on the TMDs that fit into groves on the NBDs. The conformational strain placed on the TMDs by the NBDs causes a considerable shift of the transmembrane helices, so that the substrate binding/extrusion site is made inaccessible to the cytosol and is opened to the extracellular space. Shortly thereafter, the NBDs hydrolyze and release their bound ATP, which causes them to return to the ground state, in which they push the cytosolic ends of the transmembrane domains apart. This reverses the previous conformational change in the TMDs, so that the substrate binding/extrusion site is made inaccessible to the extracellular space and opens to the cytosol.

This cycle of ATP binding and hydrolysis fuels the unidirectional motion of molecules in both ABC importers and ABC exporters; however, important structural differences between the TMDs of the importers and exporters account for their different transporting properties.

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ABC Exporters


ABC exporters serve quite diverse functions, serving notable roles as protein export machinery and efflux pumps for small molecules, such as drugs. Despite their diversity in function, ABC exporters maintain relatively strong structural similarities. All of the exporters have twelve transmembrane alpha-helices (six helices contributed by each subunit) that extend about 25 Å into the cytosol. By examining the hydrophobicity of the TMDs, it becomes clear that only the central portion of the TMD is embedded in the membrane (residues are indicated as: or ). The alpha helices contributed by each subunit do not align as parallel bundles; rather, they are considerably intertwined.

Shown here is the structure of Sav1866 from Staphylococcus aureus. Sav1866 was the first ABC exporter structure to be determined to high resolution. The structure shown here is in an ADP bound state; however, it is thought to reflect an ATP bound conformation. As expected for an ATP bound state, the ABCs are bound tightly together, and the TMDs have adopted a conformation exposing their ligand binding site to the extracellular space.

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Binding Proteins Structure


Most ABC importers are functionally dependent on high-affinity extracytoplasmic binding proteins (BPs), which are typically soluble and free-floating in the periplasm of gram-negative bacteria. In gram-positive species, the BPs are typically either covalently linked to a lipid membrane anchor, or they are bound directly to the extracytoplasmic face of the transporter.

BPs function as monomers, with each having a single ligand-binding site. Periplasmic BPs are structurally similar, each consisting of two globular lobes, dubbed the N and C domains, corresponding to the N- and C-termini. Each lobe is composed of an alpha-beta fold – alpha helices surrounding the outside of a beta sheet. The ligand-binding site is located between the two lobes, and in an unbound state, the lobes are separated, exposing the ligand-binding site to the solvent.

Ligand binding specificity is, in most cases, determined by hydrogen binding or ion-dipole interactions. Upon ligand binding, the two lobes of the BP draw closely together, desolvating the ligand and burying it within the binding cleft.

The conformational change induced by ligand binding allows the BP to interact with the transporter. Certain key residues lie to either side of the ligand-binding cleft. Upon ligand binding, these residues shift relative location, changing the nature of the transporter-binding site.

Shown here is the crystal structure of ModA, bound to tungstate. A tungstate ion is bound in the ligand binding site, where it is coordinated by an aspartate and a glutamate side chain.

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Transporter-BP Complex


Shown here is MobBC in complex with its binding protein ModA. A tungstate ion is bound in the ligand binding cleft of ModA. In this structure, the ligand binding cleft of ModBC is open to the cytosol, and the tungstate ion is prevented from entering the substrate-binding cleft of the TMD by a <scene name='User:Lori_Wetmore/Sandbox_4/Modabc/8'>gate region, which is constituted by sections of TM segments 3 and 5. The ligand-binding cleft of ModA is situated directly above the gate region of the ModB TMDs. In this crystal structure, the NBDs are not ATP bound, and thus they are in an open conformation, so that the <scene name='User:Lori_Wetmore/Sandbox_4/Modabc/9'>LSGGQ motif (pink) and the Walker A motif (yellow) are exposed to the cytosol.

In vitro studies have shown that addition of BP to importers increases their ATPase ability, especially when the substrate is also present. Interestingly, in the absence of substrate and binding protein, some type I importers display futile ATP hydrolysis – meaning that they are not actually transporting anything as they consume ATP. Other importers, however, only hydrolyze ATP when bound to their BP and while transporting substrate.

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Type I ABC Importers
<applet load='3d31' size='300' frame='true' align='left' scene='User:Lori_Wetmore/Sandbox_4/Modbc/2' target='1' caption='The molybdate/tungstate transporter was one of the first type I importers to have its structure determined to high resolution.' />

Type I importers, also referred to as the ‘small’ importers, mediate the transport of small ligands, such as ions, sugars, and amino acids. The <scene name='User:Lori_Wetmore/Sandbox_4/Modbc/6'>transmembrane domains of these transporters typically contain 12 helices (six helices contributed per subunit), with 10 helices in a core bundle. The <scene name='User:Lori_Wetmore/Sandbox_4/Modbc/5'>N-terminal helices of each subunit wrap around the outside of the partner protein’s helical bundle; however, these N-terminal helices are not present in all type I importers. For example, ModBC from Escherichia coli lacks the N-terminal helices, so its TMD contains a total of only 10 helices (five helices contributed per subunit).

Unlike in exporters, the transmembrane domains of importers are almost entirely embedded in the membrane. An examination of the <scene name='User:Lori_Wetmore/Sandbox_4/Modbc/3'>hydrophobicity of the importer TMDs reveals the extent to which the TMDs are embedded in the membrane (residues are indicated as: or ).

Due to the fact that the NBD and TMD are separate polypeptides in the case of importers, the most significant interaction between the subunits occurs at the <scene name='User:Lori_Wetmore/Sandbox_4/Modbc/4'>coupling helices.

Shown here is ModBC from Methanosarcina acetivorans, without its BP. To view interactions with the binding protein, see above.

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Type II ABC Importers
<applet load='1l7v' size='300' frame='true' align='right' scene='User:Lori_Wetmore/Sandbox_4/Btucd/2' target='1' caption='The B12 transporter is an example of type II ABC importers.' />

Shown here is the crystal structure of vitamin B12 transporter BtuCD from Escherichia coli, a good example of a type II importer. Completely assembled, this structure is 90 Å tall, 60 Å wide, and Å 30 thick. Below the TMD, there is a very large, water filled <scene name='User:Lori_Wetmore/Sandbox_4/Btucd/7'>channel that would be absent from other ABC transporters, such as exporters. It can also be observed that the two TMDs are considerably less intertwined than would be observed in the case of an exporter. The ligand channel through the center of the TMDs is lined with hydrophobic residues, provided largely by <scene name='User:Lori_Wetmore/Sandbox_4/Btucd/8'>helices 5 and 10.

Type II importers, also referred to as the ‘large’ importers, mediate the transport of larger organic compounds, such as vitamin B12 or heme. Each TMD subunit of type II importers contributes a beastly 10 <scene name='User:Lori_Wetmore/Sandbox_4/Btucd/3'>transmembrane alpha helices to the complex, so that the final structure contains 20 transmembrane helices. Interestingly, in both outward and inward facing conformations, type II importers do not appear to have specific ligand binding sites. Consequently, some speculate that type II transporters actually have little affinity for their substrates, and simply allow substrates to slide through them on conformational change. Thus, substrate specificity is almost exclusively determined by the BP, and the cleft created at the interface between the BP and the TMDs.

Much like type I importers, the TMDs of type II importers do not project very far into the cytosol, as can be determined by examining their <scene name='User:Lori_Wetmore/Sandbox_4/Btucd/4'>hydrophobicity (residues are indicated as: or ). As with type I importers, the NBDs and TMDs of type II importers are separate polypeptides that interact through <scene name='User:Lori_Wetmore/Sandbox_4/Btucd/5'>coupling helices that extend from the TMDs and fit into a cleft on the NBDs.

Although the mechanism by which ATP binding and hydrolysis is coupled to structural changes in the TMDs is presumed to be the same in type II importers as it is in other ABC transporters, to date, crystal structures have not revealed a correlation between TMD conformation and ATP binding. Thus, it is conceivable that type II transporters have a slightly different mechanism of function from the other transporters. Alternatively, some of the crystal structures determined to date may not reflect actual in vivo conformations.

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References